A bioassay technique was developed to analyze the effect of vasoactive substance(s) released from endothelial cells. Canine femoral arteries with or without endothelium were perfused with physiological salt solution at 37 degrees C. The perfusate was bioassayed with a ring of coronary artery without endothelium. A substance(s) released by the endothelial cells under basal conditions caused relaxation of unstimulated coronary arteries or relaxation of those contracted with prostaglandin F2 alpha. The release of the relaxing substance(s) was augmented by acetylcholine. The relaxation induced by acetylcholine was biphasic: an initial rapid phase followed by a partial recovery and a slowly developing prolonged relaxation; the half-life of the substance(s) causing the initial phase averaged 6.3 s. Norepinephrine, epinephrine, and ascorbic acid, given downstream of the femoral artery, reversibly prevented the second phase but only attenuated the initial relaxation. These observations indicate that an endothelium-derived relaxing substance(s) is released into the lumen of the femoral artery under basal conditions and during stimulation with acetylcholine. Catecholamines can inactivate the relaxing substance(s) but do not prevent either its production by endothelial cells or its action on vascular smooth muscle.